Evaluation of polymerase chain reaction for the detection and characterisation of rinderpest and peste des petits ruminants viruses for epidemiological studies

Abstract

The high sequence variability found in RNA viruses makes it difficult to design primers for reverse transcription-polymerase chain reaction amplification which will be certain to work with all new field isolates. To overcome this problem for the detection and differential diagnosis of rinderpest (RP) and peste des petits ruminants (PPR) viruses (V), we have designed several sets of primers, based on well-conserved sequences in the P and F genes. Analysis of a large number of field isolates from every region of the world where RPV and PPRV are found showed that no sample failed to react with more than one of the primer sets. To facilitate the multiple analyses, the reverse transcription step was performed using random hexanucleotide primers and aliquots of the cDNA were then amplified using a panel of primer sets to identify and differentiate between the virus nucleic acids in the samples. Evaluation of the method was carried out using eye swabs collected from cattle experimentally infected with RPV and goats infected with PPRV during the course of vaccine trials and on field samples such as whole blood, mouth swabs, lung, spleen and other tissues submitted to the laboratory for diagnosis. Sequencing the PCR products enabled us to examine the genetic relationships between new and previous field isolates from different geographical areas.